Method for decellularising foreign material for the production of bioprostheses

ABSTRACT

The invention relates to a method for decellularising allogenic and xenogenic foreign material for the subsequent production of bioprostheses, coated with endogenous body cells, whereby the foreign material is firstly treated in a solution of bile acid and then alcohol, each with an intermediate or subsequent rinsing step, in combination with a mechanical action on the tissue and the cells by the force generated from a flowing treatment medium, at least in the final rinsing step. After said treatment the tissue is completely rid of foreign cell material and viruses and represents an excellent starting material for the coating with cells of the bioprosthesis recipient.

[0001] The present invention relates to a method for decellularizingallogenic and xenogenic foreign material using biodetergents for theproduction of bioprostheses coated with endogenic cells of the recipientof the prosthesis.

[0002] It is required to provide an “acellular” structure that is freefrom foreign cells in order to prevent immunological reactions and toensure the growth and regeneration of the newly established endogeniccells for producing bioprostheses from allogenic and xenogenic foreignmaterial coated with endogenic cells of the later recipient. However theknown decellularization methods and uses of biodetergents fail toextract the entire cell material from the tissue matrix so that viral—asyet unknown—effects, e.g. as can be produced by viruses contained inporcine tissue, cannot be excluded.

[0003] It is the problem of the present invention to specify a methodfor decellularizing foreign material intended for being coated withendogenic cells that ensures complete but gentle removal of cells fromthe foreign tissue.

[0004] This problem is solved according to the invention by a methodcomprising the characteristics described in claim 1.

[0005] In other words, the inventive idea is to remove foreign cellsfrom the initial allogenic or xenogenic product to be re-coated withendogenic cells by combining a treatment with bile acid, a treatmentwith alcohol and upstream and downstream rinsing steps with themechanical impact of a flowing medium on the tissue matrix and the cellsto be removed at least in the last rinsing step.

[0006] The bile acid that is preferably used in the form of deoxycholicacid causes gradual—or with a mechanical impact, accelerated—coating ofthe cells with the acid to create a separating layer between the matrixmade of collagen and elastin (hereinafter called ‘collagen matrix’) andthe cell and to detach the cell from the matrix. At the same time,deoxycholic acid has a cytolytic effect. The detached cells and-thedeoxycholic acid are rinsed off in a subsequent rinsing step. Thesubsequent treatment with alcohol, preferably with ethanol or propanol,completely disposes of any residual deoxycholic acid as it dissolveswell in alcohol. The residual deoxycholic acid that may be presentdetaches any cells that remained in the matrix while the alcohol has acytocidal and antiviral effect. The subsequent last rinsing step is apreferably pulsating flow whose forces act upon the walls of therespective organ portion and expand the matrix but also apply a directmechanical force onto residual cells and remove them from the expandedmatrix.

[0007] It is conceivable that other or all steps of the method areconnected with such mechanical action by a preferably pulsating flow.Thus the pulsating deoxycholic acid flow mentioned above results infaster formation of the separating layer between the cell and thecollagen matrix due to the movement and expansion of the matrix andmakes detaching the cell easier due to the forces that act upon it.

[0008] The subclaims and the subsequent description of an embodimentdisclose other characteristics and advantageous improvements of theinvention.

[0009] Using the proposed method, it is possible to provide acellularinitial products, i.e. organ portions such as cardiac valves or vesselsthat are free from any cell material and viruses for producingbioprostheses by subsequently coating these products with endogeniccells from their respective recipient.

[0010] The apparatus for treating an organ portion consisting of aforeign material in a flowing medium includes a decellularizationchamber that receives the respective organ portion and a pump thatcreates the medium flow, both sequentially incorporated in a ring line.The ring line includes inlet and outlet valves for feeding or drainingthe respective treatment medium. The decellularization chamber can bedetached from the ring line so that said chamber and the organ portionin the medium it contains can be moved. The organ portion to be treatedis fixed and preloaded in the container by sewing it to adapters shapedlike the organ portion and placing it lengthwise in the direction offlow.

[0011] An embodiment of the invention is explained in greater detailbelow with reference to the figures. Wherein:

[0012]FIG. 1 shows an apparatus for decellularizing a cardiac valve in aflow circuit;

[0013]FIG. 2 shows a sectional view of the decellularization chamberthat is incorporated in the flow circuit and receives the cardiac valve;

[0014]FIG. 3a shows a microscopic sectional view of an aortic valve wallthat has been decellularized using the method according to theinvention; and

[0015]FIG. 3b shows a magnified view of a medial tissue section of theaortic valve wall according to FIG. 3a.

[0016] In the embodiment described here, a porcine aortic valve that wasremoved at a slaughterhouse is freed from fat, cut to size, measured,and checked for germs (fungi, aerobic and anaerobic bacteria,mycoplasma). Intermediate storage at a maximum temperature of 4° C.should not exceed seven days.

[0017] The cardiac valve prepared in this way is put into a 1% to 2%deoxycholic acid solution (or a bile acid with a similar effect) andstored therein for 24 hours at 37° C. The deoxycholic acid is capable offorming so-called adducts with a fatty acid in the form of inclusioncompounds so that the deoxycholic acid can encompass the cell on allsides, thereby dissolving its adhesive bond with the tissue matrix. Atthe same time, deoxycholic acid has a cytocidal effect.

[0018] Subsequently, a cardiac valve treated in this way is rinsed underconstant motion in a dilution set of a phosphate buffer solution (PBS)at decreasing concentrations to remove the cells treated withdeoxycholic acid from the tissue matrix.

[0019] In a subsequent third step, the cardiac valve is treated at roomtemperature for about 10 minutes in 40 per cent alcohol to produce anantiviral effect and kill any remaining cells in the collagen structure.As alcohol is a good solvent, it at the same times rinses off anyresidual acid and detaches more cells.

[0020] Using another set of a phosphate buffer solution (PBS), thecardiac valve is rinsed once again and then treated mechanically in apulsating PBS media flow. The pulsating media flow rhythmically widensthe cardiac valve that is fixed and preloaded lengthwise to the flow ina decellularization chamber and at the same time exposed to mechanicalforces. This step mechanically detaches any remaining cells from thecollagen structure so that an acellular structure is obtained from whichall cell material has been removed and which therefore cannot containany viruses. A tissue matrix of the cardiac valve treated in this waywhich is free of cells and of the decellularization media used—as shownin FIG. 2—is excellently suited for re-coating with endogenicendothelial cells from the later recipient of such a bioprosthesis, andthis bioprosthesis can be implanted into a human body without the riskof immunological reactions or viral influences.

[0021] The invention is not limited to the treatment variant describedherein, both regarding the type and origin of the foreign material usedfor producing bioprosthesis and regarding the procedural parameters aslong as the essential steps of the method, i.e. treatment with anadduct-forming bile acid and alcohol with intermediate or downstreamrinsing in combination with exposure of the respective organ portion toa preferably pulsating flow for gentle mechanical action on the tissue,are executed. The method can also be carried out by running not just thelast rinsing step but, instead or in addition, by running other or alltreatment steps in a flowing medium. This mechanically supports theeffect of the respective medium, whereby better, all-area access to thecells is achieved and the cells are easier detached or removed from theexpanded collagen matrix due to the action of the pulsating flow.

[0022] An apparatus for decellularizing a cardiac valve is shown inFIG. 1. It includes a ring line 1 that incorporates a decellularizationchamber 2 that receives the cardiac valve to be treated, a diaphragmpump 3 and a downstream equalizing chamber 4. The diaphragm pump 3 isconnected to a drive unit (not shown) via a hose line 5. An outlet valve6 and an inlet valve 7 whose functions approximately correspond to thatof a cardiac valve are integrated into the two connections of thediaphragm pump 3 to the ring line 1. The outlet valve 6 can be omittedwhen treating cardiac valves as these have valve flaps.

[0023] The core of the apparatus is the decellularization chamber 2 fordecellularizing a porcine aortic valve 8 using the additional effect offluid force. The decellularization chamber 2 consists of a transparenthollow cylinder 9 made of piacryl into the open end faces of which theteflon adapters 10 and 11 are sealingly centered and fixed, saidadapters being connected to the ring line 1 via fittings 12, 13, each ofthem comprising a fixing section 14, 15 that protrudes into the hollowcylinder 9 and has mounting holes 16, 17 radially spaced around itsperiphery for firmly holding the aortic valve 8 in a preloaded state tothe rims of the end faces. The outer diameter of the two fixing sections14, 15 of the adapters 10,11 approximately is the same as the diameterof the aortic valve 8. The rear adapter 11 can be braced via a bridge 18and a first packing 27 on the inside of a ring land 20 that is connectedto the hollow cylinder 9 by turning a threaded ring 21 whose femalethread engages in a male thread on the adapter 11. The adapter 10comprises a collar 22 that rests against the end surface of the hollowcylinder 9 and can be braced using a threaded cap 23 with a femalethread that engages in a male thread on the hollow cylinder 9. A secondpacking 19 is provided for leak proof mounting. The hose piece of thering line 1 that is topped by the decellularization chamber is made of aflexible material (silicone) to ensure flow-through due to the pulsatingpumping effect.

[0024] Due to the design and arrangement of the adapters 10, 11 asdescribed above, a suitably prepared aortic valve 8 can be sewed outsidethe hollow cylinder 9 to the opposite fixing sections 14, 15 of theadapters 10, 11. The aortic valve 8, fixed as described above, isinserted into the hollow cylinder 9. First, the deoxycholic acid isintroduced into the decellularization chamber 2 and the ring line 1 viaan inlet and outlet valve 24, 25 in the ring line 1 or one of thefittings 13, 14; then, the diaphragm pump 3 is activated so that apulsating flow of deoxycholic acid continuously flows by or through theaortic valve 8, and the mechanical force this flow exerts on the tissuecompletes the detachment and removal of cells that are foreign to therecipient of the cardiac valve. Physiological saline or phosphate buffersolution is filled into the apparatus after discharging the deoxycholicacid, and the tissue is rinsed until all the deoxycholic acids and anytoxic constituents are removed. After this rinsing step, treatment ofthe aortic valve 8 with alcohol and another rinsing step in phosphatebuffer solution follow.

[0025] All treatment steps of the decellularization method take place inthe apparatus described above in a pulsating flow of the respectivemedium. The direction of flow is the natural flow direction when thebioprosthesis is implanted. The inlet and outlet valves 24, 25 are usedfor media replacement, however fresh rinsing solution can be supplied,and used rinsing solution can be discharged, continuously for therinsing step.

[0026] It is optionally possible to carry out one or several treatmentsteps disconnected from the ring line and diaphragm pump without anymedium flowing through the decellularization chamber, which optionallymay be turned manually or using a motor, or, as stated above, to carryout individual treatment steps outside the decellularization chamber.

1. A method for decellularizing allogenic and xenogenic foreign materialusing biodetergents to produce bioprostheses coated with endogenic cellsfrom the recipient of the prosthesis, characterized in that the foreignmaterial is initially treated with bile acid to kill the cells and tocoat the cells as well as to separate the bond with the tissue matrix,then treated with alcohol to kill more cells, each of these steps beingfollowed by a rinsing step and at least the last rinsing step beingexecuted in a media flow so that the fluid forces act mechanically onthe tissue matrix and the cells.
 2. The method according to claim 1wherein the media flow is pulsating.
 3. The method according to claim 1,characterized in that a cell-encompassing deoxycholic acid is used todetach the cells with bile acid.
 4. The method according to claim 3wherein 1% to 2% deoxycholic acid is used.
 5. The method according toclaim 1 wherein 30% to 60% alcohol is used to kill the cells.
 6. Themethod according to claim 5 wherein 40% alcohol is used.
 7. The methodaccording to claim 1 wherein a phosphate buffer solution is used asrinsing medium.
 8. The method according to claim 7 wherein decreasingconcentrations of the rinsing medium are used for rinsing withnon-flowing medium.
 9. The method according to claims 1 through 7characterized in that the deoxycholic acid treatment in non-flowingmedium takes up to 24 hours, alcohol treatment takes about 20 minutes,and rinsing takes about 60 minutes.
 10. The method according to claims 1through 7 characterized in that the treatment of the foreign material indeoxycholic acid and/or alcohol and/or the intermediate rinsing cycleare carried out in a pulsating medium flow that comes close to thenatural flow conditions in the respective organ.
 11. The methodaccording to claims 1 and 10 wherein the speed of the flowing media canbe varied.
 12. The method according to claims 1, 10 and 11 characterizedin that the flowing rinsing medium is replaced continuously ordiscontinuously.
 13. The method according to claim 11, characterized inthat the concentration of the detergent flow is gradually reduced. 14.The method according to any one of claims 1 and 10 through 13,characterized in that the foreign material to be treated is held in apreloaded state in the respective media flow.
 15. An apparatus fortreating foreign material in a media flow according to claims 1 and 10through 14, characterized by a ring line (1) with a pump (3) connectedto it to generate the pulsating media flow and a decellularizationchamber (2) detachably integrated into the ring line (1) in which theallogenic or xenogenic foreign material (8) to be treated can be fixedlengthwise in a preloaded state in the direction of the treatment mediaflow using adapters (10, 11) that are detachably mounted to thedecellularization chamber (2) and comprise a fixing section (14, 15) andfittings (12, 13) for connecting the ring line (1).